CD8-expressing T cells play a critical role in eradicating intracellular parasites such as viruses. CD8+ T cells recognize MHC class I molecules in a complex with peptides of 8 to 10 residues derived from viral proteins located in the cytosol. There is tremendous interest in the mechanism by which peptides are generated. There is indirect evidence that implicates proteasomes in the generation of antigenic peptides. Proteasomes are abundant macromolecular structures present in the cytosol and nucleus in cells, and have multiple protease activities. They are thought to be responsible for energy-dependent proteolysis in which ubiquitin plays a prominent role in targeting proteins for destruction. Although it is believed that at least some proteolysis occurs in the cytosol, it is uncertain whether the ultimate determinants are generated in the cytosol or whether additional trimming occurs following transport from the cytosol. We have initiated several approaches to assess the site and nature of proteolytic mechanisms utilized in the generation of antigenic peptides. First, we have studied antigen processing in cells defective in their capacity to ubiquitinate proteins. Second, we have examined the relationship between metabolic stability of a protein and its efficiency as a source of antigenic peptides. Third, we examined the requirement for MHC-encoded proteasome subunits in antigen processing.